Hematopoietic stem cells constitute the small number of cells in the blood and bone marrow that are characterized by their ability to self-renew, and their potential to differentiate into all of the various cellular elements of the peripheral blood. Understanding of the molecular signals that confer these stem cell properties, and the mechanisms that regulate stem cell proliferation and differentiation is just emerging. Identification and characterization of these factors is critical to achieving reliable in vitro stem cell expansion and control of differentiation, with the ultimate aim of establishing clinically effective gene therapy and cellular therapy protocols. Toward this end, we are investigating the Pitx2 gene, a bicoid-related homeodomain transcription factor, which is expressed in murine hematopoietic stem/progenitor cells but not in differentiated hematopoietic cells. It is also a downstream target of the mammalian trithorax homolog gene, MLL, a critical regulator for multiple developmental programs including hematopoiesis. The central goal of this proposal is to elucidate the function of Pitx2 in hematopoietic stem cell proliferation and differentiation. Specifically, we propose to: Study the effect of loss of Pitx2 on hematopoietic cells by analyzing chimeric mice derived from embryonic stem cells that have homozygous disruption of the Pitx2 gene; Study the effect of retroviral-mediated enforced Pitx2 expression on growth and differentiation of multipotent hematopoietic cell lines, and on differentiation and engraftment of primary bone marrow cells; Test the functional role of Pitx2 as a downstream target of the MLL gene by restoring full Pitx2 activity in homozygous MLL disrupted (-/-) embryonic stem cells that are defective in their hematopoietic potentials The laboratory research will be performed by the candidate, Dr. Hui Zhang, under the guidance of a sponsor, Dr. Bernard Forget, and an advisory committee. In addition to the scientific goals, a further objective of this proposal is to serve as a vehicle for the development of the candidate into an independent and productive investigator in the area of hematopoietic stem cell biology and genetics.